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Proteasome dysfunction triggers activation of SKN-1A/Nrf1 by the aspartic protease DDI-1

  1. Nicolas J Lehrbach
  2. Gary Ruvkun  Is a corresponding author
  1. Massachusetts General Hospital, United States
  2. Harvard Medical School, United States
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Cite this article as: eLife 2016;5:e17721 doi: 10.7554/eLife.17721

Abstract

Proteasomes are essential for protein homeostasis in eukaryotes. To preserve cellular function, transcription of proteasome subunit genes is induced in response to proteasome dysfunction caused by pathogen attacks or proteasome inhibitor drugs. In Caenorhabditis elegans, this response requires SKN-1, a transcription factor related to mammalian Nrf1/2. Here, we use comprehensive genetic analyses to identify the pathway required for C. elegans to detect proteasome dysfunction and activate SKN-1. Genes required for SKN-1 activation encode regulators of ER traffic, a peptide N-glycanase, and DDI-1, a conserved aspartic protease. DDI-1 expression is induced by proteasome dysfunction, and we show that DDI-1 is required to cleave and activate an ER-associated isoform of SKN-1. Mammalian Nrf1 is also ER-associated and subject to proteolytic cleavage, suggesting a conserved mechanism of proteasome surveillance. Targeting mammalian DDI1 protease could mitigate effects of proteasome dysfunction in aging and protein aggregation disorders, or increase effectiveness of proteasome inhibitor cancer chemotherapies.

https://doi.org/10.7554/eLife.17721.001

eLife digest

Proteins perform many important roles in cells, but these molecules can become toxic if they are damaged or are no longer needed. A molecular machine called the proteasome destroys ‘unwanted’ proteins in animal and other eukaryotic cells. If the proteasome stops working properly, unwanted proteins start to accumulate and cells respond by increasing the activity of genes that make proteasomes. A protein called SKN-1 is involved in this response and activates the genes that encode proteasome proteins, but it is not understood how SKN-1 “senses” that proteasomes are not working properly.

Here, Lehrbach and Ruvkun used a roundworm called Caenorhabditis elegans to search for new genes that activate SKN-1 when the proteasome’s activity is impaired. The roundworms were genetically engineered to produce a fluorescent protein that indicates when a particular gene needed to make proteasomes is active. Lehrbach and Ruvkun identified some roundworms with mutations that cause the levels of fluorescence to be lower, indicating that SKN-1 was less active in these animals. Further experiments showed that some of these mutations are in genes that encode enzymes called DDI-1 and PNG-1. DDI-1 is able to cut certain proteins, while PNG-1 can remove sugars that are attached to proteins. Therefore, it is likely that these enzymes directly interact with SKN-1 and alter it to activate the genes that produce the proteasome.

More work is now needed to understand the details of how modifying SKN-1 changes its activity in cells. In the future, drugs that target DDI-1 or PNG-1 might be used to treat diseases in which proteasome activity is too high or low, including certain cancers and neurodegenerative diseases.

https://doi.org/10.7554/eLife.17721.002

Introduction

The proteasome is a multi-protein complex responsible for the majority of protein degradation in eukaryotic cells (Tomko and Hochstrasser, 2013). The essential function of the proteasome, and its highly conserved structure and mechanism of proteolysis renders it an attractive target for bacteria and other competitors. Production of small molecule inhibitors and protein virulence factors that target the proteasome by some bacteria and fungi exploits this vulnerability to gain a growth advantage (Fenteany et al., 1995; Groll et al., 2008; Meng et al., 1999). In addition, environmental stresses antagonize the proteasome by causing accumulation of unfolded and aggregated proteins that can form a non-productive inhibitory interaction with proteasomes (Ayyadevara et al., 2015; Deriziotis et al., 2011; Kristiansen et al., 2007; Snyder et al., 2003). Human diseases in which proteasome dysfunction is implicated highlight the importance of maintaining proteasome function in the face of these challenges (Ciechanover and Kwon, 2015; Paul, 2008; Tomko and Hochstrasser, 2013), and it follows that animal cells possess mechanisms to monitor and defend proteasome function.

A conserved response to proteasome disruption is the transcriptional up-regulation of proteasome subunit genes (Fleming, 2002; Meiners et al., 2003; Wójcik and DeMartino, 2002). In mammalian cells members of the Cap’ n’ Collar basic leucine zipper (CnC-bZip) family of stress responsive transcription factors mediate this transcriptional response. Two CnC-bZip franscription factors, Nrf1/NFE2L1 and Nrf2, have similar DNA-binding domains and may regulate an overlapping set of downstream targets. However, only Nrf1 is required for upregulation of proteasome subunits following proteasome disruption, whereas Nrf2 may activate proteasome expression under other circumstances (Arlt et al., 2009; Radhakrishnan et al., 2010; Steffen et al., 2010). The events leading to Nrf1 activation in response to proteasome disruption are complex. In vitro analyses in human and mouse cells indicate that Nrf1 is an endoplasmic reticulum (ER) membrane associated glycoprotein that is constitutively targeted for proteasomal degradation by the ER-associated degradation (ERAD) pathway. Upon proteasome inhibition Nrf1 is stabilized, undergoes deglycosylation and proteolytic cleavage, and localizes to the nucleus (Radhakrishnan et al., 2014; Sha and Goldberg, 2014; Wang, 2006; Zhang and Hayes, 2013; Zhang et al., 2015, 2007, 2014). How processing of Nrf1 is orchestrated, and its significance in responses to proteasome disruption in vivo are not understood.

Upon proteasome disruption, C. elegans induces transcription of proteasome subunit, detoxification, and immune response genes, and animals alter their behavior to avoid their bacterial food source (Li et al., 2011; Melo and Ruvkun, 2012). The transcriptional response to proteasome disruption involves skn-1, which encodes multiple isoforms of a transcription factor with similarities to both Nrf1 and Nrf2 (Blackwell et al., 2015; Li et al., 2011). skn-1 was originally identified for its essential role in embryonic development (Bowerman et al., 1992), but is also required after these early stages for stress responses in a manner analogous to mammalian Nrf1/2 (An and Blackwell, 2003; Oliveira et al., 2009; Paek et al., 2012). SKN-1 binds to the promoters of proteasome subunit genes and mediates their upregulation in response to proteasome disruption, and is required for survival of a mutant with attenuated proteasome function (Keith et al., 2016; Li et al., 2011; Niu et al., 2011). The molecular mechanism that links SKN-1 activation to the detection of proteasome dysfunction has not been established.

Here, we use genetic analysis to uncover the mechanism that couples detection of proteasome defects to these transcriptional responses in C. elegans. We find that an ER-associated isoform of SKN-1 (SKN-1A), is essential for this response. Our genetic data show that the ER-association of this transcription factor normally targets it for poteasomal degradation via ERAD, but is also required for its correct post-translational processing and activation during proteasome dysfunction. After ER-trafficking, our data argues that the PNG-1 peptide N-glycanase removes glycosylation modifications that occur in the ER, and then the DDI-1 aspartic protease cleaves SKN-1A. Each of these steps in SKN-1A processing is essential for the normal response to proteasomal dysfunction. This pathway is essential for compensation of proteasome function under conditions that partially disrupt the proteasome; when compensation is disabled, mild inhibition of the proteasome causes lethal arrest of development. Thus we reveal a vital mechanism of proteasome surveillance and homeostasis in animals.

Results

The aspartic protease DDI-1 and ERAD factors are required for transcriptional responses to proteasome disruption

The proteasome subunit gene rpt-3 is upregulated in a skn-1-dependent manner in response to proteasome disruption (Li et al., 2011). We generated a chromosomally integrated transcriptional reporter in which the rpt-3 promoter drives expression of GFP (rpt-3::gfp). This reporter gene is upregulated in response to drugs such as bortezomib or mutations that cause proteasome dysfunction. To identify the genetic pathways that sense proteasome dysfunction and trigger the activation of SKN-1, we took advantage of a regulatory allele affecting the pbs-5 locus. pbs-5 encodes the C. elegans ortholog of the beta 5 subunit of the 20S proteasome. The pbs-5(mg502) mutation causes constitutive skn-1-dependent activation of rpt-3::gfp expression, but does not otherwise alter fertility or viability (Figure 1—figure supplement 1). Following EMS mutagenesis, we isolated a collection of recessive mutations that suppress the activation of rpt-3::gfp caused by pbs-5(mg502), and identified the causative mutations by whole genome sequencing (Table 1). The collection includes multiple alleles of genes encoding factors required for ERAD. In ERAD, misfolded glycoproteins are retrotranslocated from the ER lumen to the cytoplasm, where they are degraded by the proteasome (Smith et al., 2011). We isolated 3 alleles of sel-1, a gene that encodes the C. elegans orthologue of HRD3/SEL1. HRD3/SEL1 localizes to the ER membrane and recognizes ERAD substrates in the ER (Carvalho et al., 2006; Denic et al., 2006; Gauss et al., 2006), and a single allele of sel-9, which encodes the C. elegans orthologue of TMED2/EMP24, which is also ER-localized and implicated in ER quality control (Copic et al., 2009; Wen and Greenwald, 1999). We also found mutations in png-1, which encodes the C. elegans orthologue of PNG1/NGLY1. After ERAD substrates have been retrotranslocated to the cytoplasm, PNG1/NGLY1 removes N-linked glycans to allow their degradation by the proteasome (Kim et al., 2006; Suzuki et al., 2016). Most strikingly, we isolated six alleles of C01G5.6 (hereafter ddi-1), which encodes the C. elegans orthologue of DDI1 (DNA damage inducible 1). DDI-1 is an aspartic protease, highly conserved throughout eukaryotes (Sirkis et al., 2006). DD1’s function is poorly understood, but it has been implicated in regulation of proteasome function and protein secretion (Kaplun et al., 2005; White et al., 2011).

Table 1

EMS-induced mutations that disrupt rpt-3::gfp activation.

https://doi.org/10.7554/eLife.17721.003
AlleleAffected geneEffectHomologues
mg563C01G5.6L245FDDI1. Aspartic protease.
mg555C01G5.6C277S
mg544C01G5.6G293R
mg542C01G5.6R350STOP
mg543C01G5.6M244I
mg557C01G5.6L334F
mg565sel-1G594EHRD3/SEL1. ER membrane protein, required for ERAD substrate recognition.
mg567sel-1A522T
mg547sel-1splice site
mg550sel-9S140FEMP24/TMED2. ER membrane protein.
mg561png-1G498RPNG1/NJLY1. Peptide N-glycanase. Removes N-linked glycans during ERAD.
mg564png-1splice site

We examined activation of rpt-3::gfp in ERAD and ddi-1 mutant animals following disruption of proteasome function by RNAi of the essential proteasome subunit rpt-5. rpt-5(RNAi) caused larval arrest confirming that all genotypes are similarly susceptible to RNAi. While rpt-5(RNAi) causes robust activation of rpt-3::gfp in wild-type animals, mutants lacking ERAD factors or ddi-1 failed to fully activate rpt-3::gfp (Figure 1a). The requirement for sel-11, which encodes an ER-resident ubiquitin ligase required for ERAD (Smith et al., 2011), supports a general requirement for ERAD in activation of rpt-3::gfp expression. These genes are also required for upregulation of rpt-3::gfp following proteasome disruption by bortezomib (data not shown).

Figure 1 with 1 supplement see all
ER-associated degradation factors and the aspartic protease DDI-1 are required for responses to proteasome disruption.

(a) rpt-3::gfp expression following disruption of proteasome function by rpt-5(RNAi) in various mutant backgrounds. Scale bars 100 µm. (b) Table showing growth vs. arrest phenotypes of various mutants in the presence of bortezomib or upon rpn-10 RNAi. For RNAi experiments, L1 animals were incubated for 3 days on indicated RNAi plates, and scored for developmental arrest (+; normal development, Lva; larval arrest). For bortezomib experiments, ~15 L1 animals were incubated for 4 days in liquid cultures containing varying concentrations of bortezomib, and scored for developmental progression. The number (n) of replicate bortezomib experiments performed for each genotype is shown on the right. Each colored rectangle is divided into equal parts to show results from each replicate.

https://doi.org/10.7554/eLife.17721.004

Unlike the wild type, mutants defective in ERAD, or lacking DDI-1, arrested or delayed larval development in the presence of low doses of bortezomib (Figure 1b). We analyzed independently derived alleles of png-1 and ddi-1, indicating that hypersensitivity to proteasome inhibition is unlikely to be a consequence of linked background mutations. png-1 animals consistently showed the most severe defect, and were unable to grow in the presence of very low concentrations of bortezomib. Consistent with their drug sensitivity, mild disruption of proteasome function by RNAi-mediated depletion of the non-essential proteasome subunit RPN-10 causes a synthetic larval lethal phenotype in animals mutant for png-1. The bortezomib sensitivity of ddi-1; sel-11 double mutants was not enhanced compared to that of ddi-1 single mutants, suggesting that ddi-1 and ERAD factors act in the same genetic pathway. We conclude that ERAD and DDI-1 are required for transcriptional upregulation of proteasome subunits and survival during proteasome dysfunction. Given the defective activation of rpt-3::gfp, a direct target of SKN-1, it is likely that upon proteasome disruption, these factors are required to activate SKN-1.

SKN-1A, an ER-associated isoform of SKN-1 mediates proteasome homeostasis

The skn-1 gene generates 3 protein isoforms using alternative transcription start sites (SKN-1A, SKN-1B, and SKN-1C; Figure 2a). The isoforms share an identical C-terminal DNA binding domain, but differ in their N-termini. skn-1(RNAi) targets sequences common to all three transcripts. Both skn-1(RNAi), and the skn-1(zu67) mutation cause defective responses to proteasome dysfunction (NL unpublished). skn-1(zu67) is a nonsense allele affecting an exon shared by SKN-1A and SKN-1C, but that does not affect SKN-1B, suggesting SKN-1A and/or SKN-1C are required. SKN-1C encodes a 61 kD protein expressed specifically in the intestine, and SKN-1A encodes a 71 kD protein expressed in most tissues (An and Blackwell, 2003; Bishop and Guarente, 2007; Staab et al., 2014). SKN-1A differs from SKN-1C solely by the presence of 90 additional amino acids at the N-terminus that includes a predicted transmembrane domain (Figure 2b), and SKN-1A has been found to associate with the ER (Glover-Cutter et al., 2013).

Figure 2 with 1 supplement see all
SKN-1A, a transmembrane-domain-containing isoform of SKN-1 mediates transcriptional responses to proteasome disruption.

(a,b) Schematic of the (a) skn-1 locus and (b) SKN-1A protein. In (a), the CRISPR-induced skn-1a-specific mutations are indicated. (c,d) rpt-3::gfp induction in wild type and isoform-specific skn-1 mutants in (c) the pbs-5(mg502) mutant, or (d) rpt-5(RNAi). Scale bars 100 µm. (e) Developmental arrest of isoform-specific skn-1 mutants exposed to mild proteasome disruption by rpn-10(RNAi) but not on control RNAi. Scale bar 1 mm. (f) Expression and localization of functional SKN-1A::GFP fusion protein after proteasome disruption by rpt-5(RNAi). Apparent GFP signal in control treated animals is background auto-fluorescence. Scale bar 10 µm. (g) No developmental arrest of skn-1 mutants carrying an isoform-specific skn-1a::gfp transgene, and exposed to mild proteasome disruption by rpn-10(RNAi). Scale bar 1 mm. (h) Table showing growth vs. arrest phenotypes of skn-1a mutants in in the presence of bortezomib. All skn-1a alleles are identical in their effect on skn-1a coding sequence (G2STOP). Experiments performed identically to those shown in Figure 1b, and data for the wild type from Figure 1 are shown for reference.

https://doi.org/10.7554/eLife.17721.006

We used CRISPR/Cas9 to generate an isoform-specific genetic disruption of SKN-1A, by introducing premature stop codons to the skn-1a specific exons of the skn-1 locus (hereafter referred to as skn-1a mutants). Homozygous skn-1a mutant animals are viable, and under standard conditions show a growth rate and fertility indistinguishable from the wild type. However, skn-1a mutant animals fail to activate rpt-3::gfp in the pbs-5(mg502) mutant background, or upon RNAi of essential proteasome subunit genes, or exposure to bortezomib (Figure 2c,d, data not shown). We note that in these experiments skn-1a mutants failed to activate rpt-3::gfp in all tissues, including the intestine, where SKN-1C is expressed. Consistent with the failure to upregulate rpt-3::gfp, skn-1a mutants show larval lethality when proteasome dysfunction is induced by rpn-10(RNAi) or treatment with a low dose of bortezomib (Figure 2e,h). These skn-1a mutations specifically affect SKN-1A, but leave SKN-1B and SKN-1C unaltered, indicating that SKN-1A is essential for normal responses to proteasome disruption and in the absence of SKN-1A, the other isoforms are not sufficient. A number of stimuli that trigger stabilization and nuclear accumulation of a transgenic SKN-1C::GFP fusion protein are known, but relatively little is known about whether these stimuli also affect SKN-1A (Blackwell et al., 2015). We used miniMos transgenesis (Frøkjær-Jensen et al., 2014) to generate genomically integrated single-copy transgenes that expresses C-terminally GFP-tagged full length SKN-1A (SKN-1A::GFP), and a second C-terminally GFP tagged truncated SKN-1A that lacks the DNA binding domain (SKN-1A[∆DBD]::GFP). When driven by the ubiquitously active rpl-28 promoter, we did not observe accumulation of SKN-1A::GFP or SKN-1A[∆DBD]::GFP, consistent with constitutive degradation of these fusion proteins. Upon disruption of proteasome function, we observed stabilization and nuclear localization of SKN-1A::GFP and SKN-1A[∆DBD]::GFP in many tissues (Figure 2f, Figure 2—figure supplement 1a). For unknown reasons, the SKN-1A[∆DBD]::GFP transgene accumulated to higher levels than the full length transgene (Figure 2—figure supplement 1b). We generated similar transgenes to express tagged full length and truncated SKN-1C, but did not observe any effect of proteasome disruption (data not shown). These data suggest proteasome dysfunction triggers activation of SKN-1A, but not SKN-1C.

We introduced the SKN-1A::GFP transgene into the skn-1a(mg570) and skn-1(zu67) mutant backgrounds. SKN-1A::GFP rescued the maternal effect lethal phenotype of skn-1(zu67). SKN-1A::GFP also restored wild-type resistance to proteasome disruption, as assayed by growth on rpn-10(RNAi) (Figure 3), or growth in the presence of low concentrations of bortezomib (data not shown). This indicates that the SKN-1A::GFP fusion protein is functional, and that SKN-1A::GFP is sufficient for normal responses to proteasome dysfunction even in the absence of SKN-1C (which is disrupted by the zu67 allele). As such, the transmembrane-domain-bearing SKN-1A isoform is necessary and sufficient for responses to proteasome dysfunction.

ERAD is required for constitutive SKN-1A degradation, and for activation of SKN-1A upon proteasome disruption.

(a) Western blot showing expression and post-translational processing of SKN-1A[∆DBD]::GFP in ERAD mutant animals, treated with either solvent control (DMSO) or 5 ug/ml bortezomib. SKN-1A[∆DBD]::GFP is only detected in wild-type animals upon bortezomib exposure; a major band at ~70 kD and a minor band at ~90 kD are detected. In ERAD defective mutants, the ~90 kD band is strongly detected under all conditions, and the ~70 kD band appears only following bortezomib treatment. Actin is used as a loading control. (b) Expression and localization of SKN-1A::GFP in wild type and sel-1 and sel-11 ERAD defective mutants after proteasome disruption by rpt-5(RNAi). In ERAD defective mutants, SKN-1A::GFP fails to localize to the nucleus. Scale bar 10 µm. (c) Expression and localization of SKN-1A::GFP in wild type and png-1 mutants after proteasome disruption by rpt-5(RNAi). In png-1 mutants, SKN-1A::GFP is able to localize to the nucleus, although at reduced levels compared to the wild-type. Scale bar 10 µm.

https://doi.org/10.7554/eLife.17721.008

Mutation of ddi-1 does not enhance the sensitivity of skn-1a mutants to bortezomib, suggesting that DDI-1 acts through SKN-1A to promote resistance to proteasome inhibitors (Figure 2h). Additionally removing SEL-11 weakly enhanced the bortezomib sensitivity of ddi-1 skn-1a double mutants, and also caused occasional growth defects even in the absence of proteasome disruption, suggesting that ERAD promotes resistance to proteasome inhibitors largely, but not solely, through regulation of SKN-1A. We examined how ERAD factors regulate SKN-1A using the SKN-1A::GFP transgenes. sel-1 and sel-11 mutants accumulate high levels of SKN-1A[∆DBD]::GFP even in the absence of proteasome inhibitors, showing that SKN-1A is constitutively targeted for proteasomal degradation via ERAD (Figure 3a). Upon proteasome disruption, sel-1 and sel-11 mutants show defects in SKN-1A::GFP nuclear localization consistent with defective release from the ER (Figure 3b). Following proteasome disruption in png-1 mutants SKN-1A::GFP localizes to the nucleus, indicating PNG-1 acts downstream of release from the ER (Figure 3b). Lower levels of SKN-1A::GFP accumulate in the nuclei of png-1 mutants than in the wild type, but this mild effect is unlikely to fully account for the severely defective responses to proteasome inhibition in png-1 mutant animals, suggesting retention of glycosylation modifications normally removed by PNG-1 likely disrupts SKN-1A’s nuclear function. These data suggest that activation of ER-associated and N-glycosylated SKN-1A is required for responses to proteasome dysfunction.

DDI-1 aspartic protease localizes to both nucleus and cytoplasm, and is upregulated upon proteasome disruption

To examine the expression and subcellular localization of the DDI-1 protease, we used miniMos to generate a single copy integrated transgene expressing full length DDI-1 fused to GFP at the N-terminus, under the control of the ddi-1 promoter. The GFP::DDI-1 fusion protein is expressed in most tissues and shows diffuse cytoplasmic and nuclear localization under control conditions, and can rescue a ddi-1 mutant (see below). Following disruption of proteasome function by rpt-5(RNAi), GFP::DDI-1 expression is dramatically induced, and GFP::DDI-1 is enriched in nuclei (Figure 4a). We used CRISPR/Cas9 to modify the ddi-1 locus to incorporate an HA epitope tag near the N-terminus of endogenous DDI-1. Following bortezomib treatment of ddi-1(mg573[HA::ddi-1]) animals, we observed strong upregulation (greater than 10-fold, based on blotting of diluted samples) of the HA-tagged endogenous DDI-1 (Figure 4b). The ddi-1 promoter contains a SKN-1 binding site (Niu et al., 2011). Upregulation of GFP::DDI-1 by rpt-5(RNAi) is greatly reduced in skn-1a(mg570) mutants (Figure 4c), suggesting that DDI-1 upregulation is mostly mediated by SKN-1A. The remaining weaker ddi-1 upregulation in the skn-1a mutant may represent a second skn-1a-independent mechanism that couples DDI-1 levels to proteasome function.

DD1-1 is upregulated upon proteasome disruption.

(a) A functional GFP::DDI-1 fusion protein is strongly induced and localizes to the nucleus upon proteasome disruption by rpt-5(RNAi). Scale bar 20 µm. (b) Western blot showing induction of HA-tagged endogenous DDI-1 upon proteasome disruption by bortezomib. Actin is used as a loading control. (c) Induction of GFP::DDI-1 upon proteasome disruption by rpt-5(RNAi) is lost in skn-1a mutants. Scale bar 20 µm.

https://doi.org/10.7554/eLife.17721.009

DDI-1 is required for proteolytic cleavage of SKN-1A downstream of ER trafficking

The EMS-induced ddi-1 missense alleles that cause failure to activate rpt-3::gfp are clustered within the aspartic protease domain of DDI-1, and affect conserved residues that are thought to form the substrate-binding pocket of the enzyme (Figure 5a,b), suggesting that the protease activity of DDI-1 is required (Sirkis et al., 2006). We used CRISPR/Cas9 mutagenesis to generate a protease dead mutant containing two amino acid substitutions at conserved residues of the catalytic motif, including the aspartic acid residue that forms the active site (D261N, G263A). We additionally isolated a CRISPR-induced deletion that deletes most of the aspartic protease domain and introduces a frameshift, which we presume to be a null allele. Both mutations cause a similar, strong defect in rpt-3::gfp activation by the pbs-5(mg502) mutant, or upon proteasome RNAi, and cause a similar sensitivity to bortezomib (Figure 5c, data not shown).

Figure 5 with 1 supplement see all
The DDI-1 aspartic protease is required for proteolytic activation of SKN-1A.

(a) Schematic of the DDI-1 protein showing residues affected by EMS-induced loss of function alleles. (b) Multiple alignment of the aspartic protease domain of DDI-1. Red text indicates residues affected by mutations that disrupt rpt-3::gfp activation, blue text indicates the DS/TGAQ catalytic motif. (c) rpt-3::gfp induction in the pbs-5(mg502) mutant background (left panel), compared to animals carrying a ddi-1 deletion (middle panel), or a point mutation affecting the catalytic motif (right panel). Scale bars 100 µm. (d) Localization of SKN-1A::GFP in wild-type and ddi-1 mutant animals following disruption of proteasome function by rpt-5(RNAi). SKN-1A::GFP nuclear localization is intact in the ddi-1 mutant. In some gut cells of ddi-1 mutant animals SKN-1A::GFP localizes to puncta that are not detected in the wild type. Scale bars 10 µm. (e) Localization of SKN-1A[∆DBD]::GFP in gut nuclei of wild-type and ddi-1 mutant animals. Nuclear foci of SKN-1A[∆DBD]::GFP are found in ddi-1 mutants, but not wild type. (f) Western blot showing expression and processing of SKN-1A[∆DBD]::GFP in ddi-1 mutant animals, treated with either solvent control (DMSO) or 5 ug/ml bortezomib, and blotted for GFP. In the ddi-1 mutant animals, the major band detected is ~30 kD larger than in the wild type. (g) Western blot showing expression and processing of HA::SKN-1A:GFP in ddi-1 mutants animals, treated with either solvent control (DMSO) or 5 ug/ml bortezomib, and blotted for HA. In the wild type, a ~20 kD band is detected in animals exposed to bortezomib. In ddi-1 mutants this low molecular weight fragment is absent, and a ~110 kD band is detected. In (f) and (g) ddi-1 mutations were ddi-1(mg571)[deletion] or ddi-1(mg572)[active site] and actin is used as a loading control.

https://doi.org/10.7554/eLife.17721.010

S. cervisiae Ddi1 contains an N-terminal ubiquitin-like (UBL) domain and a C-terminal ubiquitin-associated (UBA) domain, but these domains are not detected by standard protein sequence comparisons with C. elegans DDI-1. To address the possibility that UBL or UBA domains with highly divergent sequence may be present in DDI-1, we generated N-terminally truncated (∆N), and C-terminally truncated (∆C) gfp::ddi-1 transgenes. We tested their ability to rescue the bortezomib sensitivity phenotype of ddi-1(mg571) alongside wild-type gfp::ddi-1 and an aspartic protease active site (D261N) mutant. The active site mutation abolished rescue by the gfp::ddi-1 transgene, whereas the ∆N and ∆C truncated transgenes restored bortezomib sensitivity to near wild-type levels (Figure 5—figure supplement 1). These data are consistent with the lack of conservation of the UBL and UBA domains of DDI-1, and confirm the essential role of DDI-1 aspartic protease activity.

In animals lacking DDI-1, SKN-1A::GFP localizes at normal levels to the nucleus upon proteasome disruption by rpt-5(RNAi), suggesting that DDI-1 regulates SKN-1A function after nuclear localization of the transcription factor (Figure 5d). We noticed that SKN-1A::GFP occasionally showed abnormal localization within gut nuclei of ddi-1 mutants, accumulating in highly fluorescent puncta. We observed this defect for both SKN-1A::GFP and SKN-1A[∆DBD]::GFP, indicating that the DBD of SKN-1A is not required for this mis-localization (Figure 5e).

As in the wild type, SKN-1A[∆DBD]::GFP does not accumulate in the absence of proteasome disruption in ddi-1 mutants, indicating that the DDI-1 peptidase does not participate in constitutive degradation of SKN-1A by the proteasome (Figure 5f). SKN-1A[∆DBD]::GFP accumulates to similar levels upon proteasome disruption by bortezomib in wild-type and ddi-1 mutants, but in ddi-1 mutants is ~20 kD larger than in the wild type, and approximates the expected size of SKN-1A[∆DBD]::GFP. To test whether these differences reflect DDI-1-dependent proteolytic processing of SKN-1A, we generated a transgene that expresses full length SKN-1A with an N-terminal HA tag and a C-terminal GFP tag (HA::SKN-1A::GFP). The expression, localization and rescue activity of the dually tagged fusion protein is indistinguishable from that of the full length SKN-1A::GFP transgene. In wild-type animals carrying the HA::SKN-1A::GFP transgene, Western blotting for the HA tag reveals a ~20 kD band that accumulates specifically upon proteasome disruption by bortezomib treatment. In ddi-1 deletion or active site mutants, a ~110 kD protein accumulates upon proteasome disruption, equivalent in size to full-length HA::SKN-1::GFP (Figure 5g). As such, SKN-1A is cleaved at a position approximately 20 kD from the N-terminus, and the protease active site of DDI-1 is required for this cleavage.

In sel-1 and sel-11 mutants, unprocessed HA::SKN-1A::GFP is present in both control and bortezomib treated animals, with some processed HA::SKN-1A::GFP appearing upon bortezomib treatment (Figure 6a). In png-1 mutant animals HA::SKN-1A::GFP is incompletely processed following proteasome disruption (Figure 6b), suggesting that DDI-1 dependent cleavage of SKN-1A normally occurs after ER trafficking and deglycosylation. Further, in wild-type animals, an HA::SKN-1A::GFP transgene that lacks the putative transmembrane domain is not targeted for constitutive degradation, and is not subject to detectable proteolytic cleavage upon proteasome disruption (HA::SKN-1A[∆TM]::GFP; Figure 6c). Although expressed, HA::SKN-1A[∆TM]::GFP is unable to rescue the bortezomib sensitivity of skn-1a mutants (data not shown). These results indicate that ER trafficking is needed to target SKN-1A for later cleavage and activation by ddi-1.

Figure 6 with 1 supplement see all
Proteolytic processing of SKN-1A occurs downstream of ER trafficking.

(a) Western blot showing expression and processing of HA::SKN-1A::GFP upon proteasome disruption by bortezomib in sel-1 and sel-11 mutant animals. ERAD-defective mutants accumulate similar levels of uncleaved (~110 kD) HA::SKN-1A::GFP in both absence and presence of bortezomib. A relatively small amount of the ~20 kD cleavage product accumulates only upon exposure to bortezomib. (b) Western blots comparing expression and processing of HA::SKN-1A::GFP upon proteasome disruption by bortezomib in wild-type and png-1 mutant animals. Both cleaved (~20 kD) and uncleaved (smear ~100-150 kD) HA::SKN-1A::GFP accumulates upon bortezomib treatment in png-1 mutants. (c) Western blot showing expression and processing of HA::SKN-1A[∆TM]::GFP upon proteasome disruption by bortezomib, in otherwise wild-type animals. Uncleaved (~100 kD) HA::SKN-1A[∆TM]::GFP is detected at similar levels under both conditions suggesting the protein is not subject to proteasomal degradation, and a low molecular weight cleavage product is not detected under either condition. For each experiment, mixed stage cultures were treated with either solvent control (-), or 5 ug/ml bortezomib (+) prior to collection for SDS-PAGE. HA::SKN-1A::GFP/ HA::SKN-1A[∆TM]::GFP is detected by anti-HA antibodies, Actin is used as a loading control.

https://doi.org/10.7554/eLife.17721.012

Discussion

Our genetic screen for mutants that fail to activate SKN-1 and dissection of the isoform-specific role of SKN-1A reveals the molecular details of proteasome surveillance. We show that SKN-1A is an ER associated protein that is normally targeted by the ERAD pathway for proteasomal degradation in the cytoplasm. Mutations affecting ERAD genes sel-1/SEL1/HRD3 and sel-11/HRD1, stabilize SKN-1A, but also disrupt localization of SKN-1A following proteasome disruption, due to a failure to efficiently release SKN-1A from the ER. Our data argues that following release from the ER, SKN-1A must be deglycosylated by PNG-1 and cleaved by DDI-1 to become fully active Figure 6—figure supplement 1.

The bortezomib sensitivity defect of png-1 mutants is similar in strength to skn-1a mutants, and both png-1 and skn-1a mutations are synthetic lethal with rpn-10(RNAi). These similarities suggest that SKN-1A activity may be completely abolished in the absence of PNG-1; likely as a result of failure to deglycosylate SKN-1A following its retrotranslocation from the ER, although we cannot rule out the possibility that deglycosylation of other proteins contributes indirectly to SKN-1A function. Surprisingly, we were unable to generate png-1; skn-1a double mutants, apparently due to lethality of double mutant embryos (NL unpublished). This indicates that in png-1 mutant animals, SKN-1A (likely in a glycosylated state) retains a function that is essential for development. This suggests that SKN-1A is not only important for proteasome homeostasis, but is also important for cellular homeostasis upon disruption of glycoprotein metabolism. Consistently, skn-1 mutants are hypersensitive to tunicamycin, an inhibitor of protein glycosylation (Glover-Cutter et al., 2013).

NGLY1, the human PNG-1 orthologue, plays important roles in human development; NGLY1 deficiency, a recently described genetic disorder of protein deglycosylation, is caused by mutations at the NGLY1 locus (Enns et al., 2014). Failure to deglycosylate Nrf1, and consequent defects in proteasome homeostasis, likely contributes to the symptoms associated with NGLY1 deficiency. As such our work identifies a pathway that may be targeted for treatment of NGLY1 deficiency - genetic screens for suppressors of the defective proteasome gene regulation of png-1 mutants could indicate targets for drug development.

The expression of DDI-1 is dramatically responsive to proteasome inhibition, indicating that its synthesis or stability is coupled to surveillance of proteasome dysfunction. ChIP analysis of SKN-1 shows binding at the promoter element of the DDI-1 operon (Niu et al., 2011), suggesting that DDI-1 upregulation may occur via SKN-1 mediated transcriptional regulation, and we observed a defect in the upregulation of GFP::DDI-1 following proteasome disruption in skn-1a mutants. This suggests a positive feedback loop, wherein SKN-1A upregulates DDI-1, and DDI-1 promotes SKN-1A activation. Positive feedback may ensure a timely and robust response to proteasome inhibition.

Mutation of ddi-1 causes defective regulation of rpt-3::gfp and increases sensitivity of C. elegans to proteasome inhibitors. SKN-1A is cleaved at a site ~20 kD from the N-terminus, and ddi-1 is required for this cleavage. The requirement for its catalytic function strongly suggests that DDI-1 is the enzyme directly responsible for cleavage of SKN-1A, although we cannot rule out the possibility that DDI-1 acts upstream of an as-yet unidentified second protease. There are precedents for cascades of proteases, for example caspases in apoptosis, complement cascades in immunology, and thrombin cascades in blood clotting. Our genetic screen for proteasome surveillance defective mutants isolated six independent ddi-1 alleles, but as yet no alleles of any other genes that encode proteases. This argues that either DDI-1 is the only protease in the pathway, or that any other proteases function redundantly or have other essential functions.

Uncleaved SKN-1A localizes to the nucleus in ddi-1 mutants, so cleavage of SKN-1A is not essential for its nuclear localization, and SKN-1A cleavage may occur either in the nucleus or cytoplasm. Given that GFP::DDI-1 is largely (but not exclusively) nuclear under conditions of proteasome disruption, we speculate that DDI-1 cleavage of SKN-1A is nuclear. In either case, unusually for a membrane-associated transcription factor, SKN-1A is released from the ER by a mechanism that does not require proteolytic cleavage. DDI-1-dependent cleavage therefore activates SKN-1A by some other mechanism(s) downstream of ER release. Cleavage of SKN-1A may be required to remove domains in the N-terminus that interfere with its normal function in the nucleus. For example, retention of the hydrophobic transmembrane domain may be disruptive once the protein has been extracted from the ER membrane.

Mutant SKN-1A lacking the transmembrane domain is not subject to proteasomal degradation, and is not cleaved by DDI-1. So, in addition to serving to link SKN-1A levels to proteasome function via ERAD, ER trafficking of SKN-1A is important for subsequent DDI-1-dependent activation. The bortezomib sensitivity of skn-1a mutants (or skn-1a mutants carrying the transmembrane domain-lacking transgene) is more severe than that of ddi-1 and ERAD mutants, so ER-association must also promote SKN-1A activation by additional mechanisms. As well as proteasome disruption, skn-1 is implicated in responses to several endocrine and environmental stimuli (Blackwell et al., 2015). Modifications such as glycosylation that SKN-1A acquires in the ER may tailor its activity to respond to proteasome dysfunction, identifying these modifications and how they are regulated will be of interest.

S. cerevisiae Ddi1 contains an N-terminal UBL domain and a C-terminal UBA domain, This domain architecture is typical of extraproteasomal ubiquitin receptors, which play a role in recruiting ubiquinated proteins to the proteasome (Tomko and Hochstrasser, 2013). S. cerevisiae Ddi1 binds to both ubiquitin and the proteasome, and participates in the degradation of some proteasome substrates (Bertolaet et al., 2001; Gomez et al., 2011; Kaplun et al., 2005; Nowicka et al., 2015), and synthetic genetic interactions with extraproteasomal ubiquitin receptor and proteasome subunit mutants supports a role for Ddi1 in proteasome function (Costanzo et al., 2010; Díaz-Martínez et al., 2006). Although the aspartic protease domain is highly conserved, DDI-1 in C. elegans and related nematodes apparently lacks both UBL and UBA domains, and the UBA domain is not found in mammalian Ddi1 orthologues, so it remains unclear whether Ddi1 orthologues function as extraproteasomal ubiquitin receptors in animals (Nowicka et al., 2015). The effect of ddi-1 on development upon proteasome disruption by bortezomib is entirely dependent on skn-1a, indicating that DDI-1 promotes resistance to proteasome disruption via SKN-1A, rather than a general effect on proteasome function. Regardless, it will be of interest to determine whether DDI-1 binds to proteasomes and/or ubiquitin, and whether this affects its function in SKN-1A activation.

Activation of the mammalian SKN-1 homologue Nrf1 involves both deglycosylation and proteolytic cleavage, but the enzymes responsible are not known (Radhakrishnan et al., 2014; Zhang et al., 2015). However, a large scale screen for gene inactivations that render cells more sensitive to proteasome inhibitors supports a model that human DDI1 protease also processes Nrf1: DDI2 (one of two human orthologues of DDI-1) and Nrf1 were highly ranked hits in this screen that identified hundreds of gene inactivations that increase sensitivity of multiple myeloma cells to proteasome inhibitors (Acosta-Alvear et al., 2015). This suggests that DDI2 is required to cleave and activate Nrf1 in human cells. The site at which Nrf1 is cleaved during proteasome dysfunction has been identified (Radhakrishnan et al., 2014), but the primary sequence of this site is not conserved in SKN-1A. Comparisons of SKN-1A with its nematode orthologues reveals conservation at positions consistent with the ~20 kD cleavage product we have observed (NL unpublished). It is possible that DDI-1 and its substrate(s) have divergently co-evolved in different lineages. Thus, we suggest that DDI-1 and SKN-1A are core components of a conserved mechanism of proteasome surveillance in animals.

Here we have shown that correct post-translational processing of SKN-1A is essential for development if proteasome function is disrupted. Deregulated proteasome function a feature of aging and age-related disease (Saez and Vilchez, 2014; Taylor and Dillin, 2011). skn-1 is a critical genetic regulator of longevity, and controls lifespan in part through regulation of proteasome function (Blackwell et al., 2015; Steinbaugh et al., 2015) As such, the SKN-1A processing pathway described here suggests the mechanism that links SKN-1/Nrf to proteasome function and longevity.

Proteasome inhibitors are important drugs in the treatment of multiple myeloma, but relapse and emergence of drug resistant tumors remains a challenge (Dou and Zonder, 2014). Nrf1 promotes survival of cancerous cells treated with proteasome inhibitors, and activation of this pathway might mediate resistance (Acosta-Alvear et al., 2015; Radhakrishnan et al., 2010; Steffen et al., 2010). Blocking the activation of proteasome subunit gene expression by Nrf1 has been proposed as a potential strategy to improve effectiveness of proteasome inhibitors in cancer treatment. The conserved SKN-1A/Nrf1 processing factors we have identified, particularly DDI-1, are ideal targets for such an approach.

Materials and methods

C. elegans maintenance and genetics

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C. elegans were maintained on standard media at 20°C and fed E. coli OP50. A list of strains used in this study is provided in Supplementary Table 1. Mutagenesis was performed by treatment of L4 animals in 47 mM EMS for 4 hr at 20°C. RNAi was performed as described in Kamath and Ahringer (2003). The mgIs72[rpt-3::gfp] integrated transgene was generated from sEx15003 (Hunt-Newbury et al., 2007), using EMS mutagenesis to induce integration of the extrachromosomal array. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). sel-11(tm1743) was kindly provided by Shohei Mitani. png-1(ok1654) was generated by the C. elegans Gene Knockout Project at the Oklahoma Medical Research Foundation, part of the International C. elegans Gene Knockout Consortium.

Identification of EMS induced mutations by whole genome sequencing

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Genomic DNA was prepared using the Gentra Puregene Tissue kit (Qiagen, #158689) according to the manufacturer’s instructions. Genomic DNA libraries were prepared using the NEBNext genomic DNA library construction kit (New England Biololabs, #E6040), and sequenced on a Illumina Hiseq instrument. Deep sequencing reads were analyzed using Cloudmap (Minevich et al., 2012).

Following deep sequencing analysis, a number of criteria were taken into account to identify the causative alleles, as shown in Supplemental file 1. In many cases, the causative alleles were strongly suggested by the identification of multiple independent alleles for a given gene. Even for those genes only identified by a single allele, the strong functional connection with other independently mutated genes suggests that they are causative (e.g. isolation of multiple alleles of the sel gene class). We also obtained genetic linkage data supporting these assignments for most alleles. For most of the mutants considered, deep sequencing was performed using a DNA from a pool of 20–50 mutant F2s generated by outcrossing the original mutant strain to the parental (non-mutagenised) background, which allowed us to use Cloudmap variant discovery mapping to identify the genetic linkage of the causative allele, or linkage was confirmed by testing linkage in crosses with strains carrying precisely mapped miniMos insertions. For ddi-1 and png-1, we confirmed that disruption by an independent means (with an independently derived allele) has the same effect on rpt-3::gfp expression as the EMS-induced mutation.

Identification of the pbs-5(mg502) mutation

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The mg502 allele was isolated in an EMS mutagenesis screen in which mgIs72[rpt-3::gfp] animals were screened for recessive mutations causing constitutive activation of GFP expression. The mutation was identified as described above. The pbs-5(mg502) lesion is a 122bp deletion in the promoter of CEOP1752. This operon consists of K05C4.2 and pbs-5. Animals carrying this mutation show constitutive activation of rpt-3::gfp, but have normal growth and fertility under control conditions.

Genome modification by CRISPR/Cas9

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Guide RNAs were selected by searching the desired genomic interval for ‘NNNNNNNNNNNNNNNNNNRRNGG’, using Ape DNA sequence editing software (http://biologylabs.utah.edu/jorgensen/wayned/ape/). All guide RNA constructs were generated by Q5 site directed mutagenesis as described (Dickinson et al., 2013). Repair template oligos were designed as described (Paix et al., 2014; Ward, 2015). Injections were performed using the editing of pha-1 (to restore e2123ts) or dpy-10 (to generate cn64 rollers) as phenotypic co-CRISPR markers (Arribere et al., 2014; Ward, 2015). Injection mixes contained 60 ng/ul each of the co-CRISPR and gene of interest targeting Guide RNA/Cas9 construct, and 50 ng/ul each of the co-CRISPR and gene of interest repair oligos. Guide RNA and homologous repair template sequences are listed in Supplemental file 1.

Transgenesis

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Cloning was performed by isothermal/Gibson assembly (Gibson et al., 2009). All plasmids used for transgenesis are listed in Supplemental file 1. All miniMos constructs were assembled in pNL43, a modified version of pCFJ909 containing the pBluescript MCS, and are described in more detail below. MiniMos transgenic animals were isolated as described, using unc-119 rescue to select transformants (Frøkjaer-Jensen et al., 2014).

MiniMos constructs

skn-1 constructs

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To generate constructs that express specific SKN-1 isoforms, we cloned skn-1c and skn-1a ‘minigenes’. The skn-1c minigene was assembled from 3 genomic fragments from the skn-1 locus: (1) skn-1c exon 1, (2) skn-1c exons 2 & 3, (3) skn-1c exons 4, 5 & 6. These three fragments were fused in frame to generate a skn-1c coding sequence lacking the two largest introns that may contain regulatory information. The skn-1a minigene was assembled by fusing two additional fragments, corresponding to the first two exons of skn-1a, to the 5’ end of the skn-1c minigene, eliminating the large intron that contains the presumed skn-1c promoter. These minigenes were then fused in frame with an N-terminal HA tag and/or a C-terminal GFP tag, and inserted into pNL43 with the rpl-28 promoter (605 bp immediately upstream of the rpl-28 start codon) and tbb-2 3’UTR (376 bp immediately downstream of the tbb-2 stop codon). skn-1a[∆DBD]::gfp and skn-1c[∆DBD]::gfp were generated by deleting sequence corresponding to the final 94 amino acids from the each construct (i.e. amino acids 530–623 of SKN-1A and amino acids 440–533 of SKN-1C) by isothermal assembly using appropriate fragments of the respective minigenes. skn-1a[∆TM]::gfp was generated by deleting sequence corresponding to amino acids 39–59 of SKN-1A, by isothermal assembly of appropriate fragments of the skn-1a minigene.

ddi-1 constructs

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The genomic C01G5.6/ddi-1 coding sequence was fused in frame with GFP at the N-terminus. The gfp::ddi-1 fragment was inserted into pNL43 with the ddi-1 promoter (803 bp immediately upstream of the start codon), and the tbb-2 3’UTR. The gfp::ddi-1[D261N] construct was generated by site-directed mutagenesis, and the N- and C-terminal truncation constructs were generated by isothermal assembly using appropriate fragments of the ddi-1 genomic coding sequence.

Microscopy

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Low magnification bright field and GFP fluorescence images (those showing larval growth and rpt-3::gfp expression) were collected using a Zeiss AxioZoom V16, equipped with a Hammamatsu Orca flash 4.0 digital camera camera, and using Axiovision software. High magnification differential interference contrast (DIC) and GFP fluorescence images (those showing SKN-1A::GFP and GFP::DDI-1 expression) were collected using a Zeiss Axio Image Z1 microscope, equipped with a Zeiss AxioCam HRc digital camera, and using Axiovision software. Images were processed using ImageJ software. For all fluorescence images, any images shown within the same figure panel were collected together using the same exposure time and then processed identically in ImageJ.

Bortezomib sensitivity assays

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Bortezomib sensitivity was assessed by the ability of L1 animals to develop in the presence of a range of concentrations of bortezomib (LC Laboratories, #B1408). The assays were carried out in liquid culture in ½ area 96 well plates (Corning, #3882). Each well contained a total volume of 35uL. We mixed ~15 L1 larvae with concentrated E. coli OP50 suspended in S-basal (equivalent to bacteria from ~200 uL of saturated LB culture), supplemented with 50 ug/ml Carbenicillin, and the desired concentration of bortezomib. All treatment conditions contained 0.01% DMSO. The plates were sealed with Breathe-Easy adhesive film (Diversified Biotech, #9123–6100). The liquid cultures were incubated for 4 days at 20°C and then C. elegans growth was manually scored under a dissecting microscope. Growth was scored into three categories: (1) Normal - indistinguishable from wild type grown in DMSO control, most animals reached adulthood; (2) Delayed development - most animals are L3 or L4 larvae; (3) Larval arrest/lethal - all animals are L1 or L2 larvae. For each genotype all conditions were tested in at least 2 replicate experiments.

Western blot following bortezomib treatment

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Drug treatments were performed in liquid culture in 6-well tissue culture plates. In each well we mixed C. elegans suspended in S-basal (~1000–2000 worms collected from a mixed stage culture grown at 20°C on NGM agar plates) and E. coli OP50 in S-Basal (equivalent to E. coli from ~4 mL saturated LB culture), supplemented with 50 ug/ml Carbenicillin, and the desired concentration of bortezomib, and made the mixture up to a final volume of 700 ul. All wells contained 0.01% DMSO. The tissue culture plates were sealed with Breathe-Easy adhesive film and incubated at 20°C for 7–9 hr. After the treatment the animals were collected to 1.5 ml microcentrifuge tubes, washed twice in PBS to remove bacteria and the worm pellet was snap frozen in liquid nitrogen and stored at −80°C. The worm pellet was briefly thawed on ice, mixed with an equal volume of 2x Sample buffer (20% glycerol, 120 mM Tris pH 6.8, 4% SDS, 0.1 mg/ml bromophenol blue, 5% beta mercaptoethanol), heated to 95°C for 10 min, and centrifuged at 16,000g for 10 min to pellet debris. SDS-PAGE and western blotting was performed using NuPAGE apparatus, 4–12% polyacrylamide Bis-Tris pre-cast gels (Invitrogen, #NP0321) and nitrocellulose membranes (Invitrogen, #LC2000) according to the manufacturer’s instructions. The following antibodies were used: mouse anti-GFP (Roche; #11814460001); HRP-conjugated mouse anti-HA (Roche, # 12013819001), mouse anti-Actin (Abcam; #3280).

Multiple alignment of protein sequences

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Multiple alignment was performed using Clustal Omega (www.ebi.ac.uk/tools/clustalo).

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Decision letter

  1. Andrew Dillin
    Reviewing Editor; Howard Hughes Medical Institute, University of California, Berkeley, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Proteasome dysfunction triggers activation of SKN-1A/Nrf1 by the aspartic protease DDI-1" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom, Andrew Dillin, is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Kevin Struhl as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Andrew Dillin (Reviewing Editor and Reviewer #1) and Raymond J Deshaies (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. As you find within the reviews, each reviewer was very enthusiastic about this body of work. While we each had minor comments and questions, we believe the body of work is a significant advance for the field. We ask that you provide a response to each comment from the reviewers.

Reviewer #1:

In this body of work Ruvkun and colleagues begin to query proteasome regulation using elegant genetics and the nematode C. elegans. In this data packed manuscript they outline the role of DDI-1, an aspartic protease, that moves to the nucleus under conditions of proteasome stress. The protease domain is more than likely required to regulate a single isoform of skn-1, thereby regulating its nuclear activities. Throughout there is great genetic interaction studies between ddi-1, skn-1 and components of the ERAD pathway (which are required to liberate skn-1 from the ER). There is a cornucopia of findings throughout and almost every stone has been turned.

Minor comments:

The western bot shown in Figure 4A is blown out. It’s ok to show it, but the authors should also include a lighter exposed one.

It would also help the reader to include a model describing skn-1 and ddi-1 under static vs. proteasome stress.

The induction of rapt is followed by GFP reporters. Does the endogenous gene also show similar regulation?

Lastly, the movement of ddi-1 to the nucleus is shown during proteasome stress. Do other stresses also regulate its movement?

Reviewer #2:

In this manuscript, Lehrbach and Ruvkun report the identification of C. elegans ddi-1 as a gene that is required for the activation of proteasome gene transcription in response to chemical inhibition of the proteasome or knockdown of proteasome subunits. It was shown over 10 years ago that inhibition of the proteasome in mammalian cells results in transcriptional induction of genes that encode proteasome subunits. Six years ago, the Deshaies group showed that this induction requires Nrf1 in mouse cells, and this was followed by a report from the Kruger group that the Nrf1 ortholog TCF11 mediates the response in human cells. They also showed that TCF11 is a substrate for the ERAD pathway. More recently, the Deshaies group reported the surprising observation that most of Nrf1 enters the ER lumen during its biogenesis, but is quickly retrotranslocated to the cytosol in p97-dependent manner, where it is clipped on the C-terminal side of tryptophan-103. This retrotranslocation and clipping were shown to be essential for Nrf1 activation. However, the protease was not identified, although a subsequent report from Fred Goldberg's group claimed that Nrf1 processing is carried out by partially inhibited proteasomes.

Lehrbach and Ruvkun carried out a screen to identify genes required for activation of the C. elegans ortholog of Nrf1, SKN-1A. They identify DDI-1, which is an aspartyl protease of largely unknown function, that in budding yeast doubles as a substrate receptor for the proteasome. They also identify ERAD genes including PNG-1, which is the C. elegans ortholog of the protein N-glycosidase enzyme NGLY1 that cleaves asparagine-linked oligosaccharides from retrotranslocated proteins. Overall, this is a compelling manuscript that makes an important finding that is likely to have broad ramifications not only for Nrf1 and protein homeostasis, but also for aging, neurodegeneration, and possibly cancer therapy. In my view, this manuscript should be an exceptionally high priority for eLife.

I have several comments, which are all modest in nature:

1) At the outset of Results, it should be explained what pbs-5(mg502) is, and what is the rationale for its use. I had to do a google search to figure out it is a mutation in a 20S proteasome subunit.

2) What happens in a png-1 ERAD double mutant in terms of growth in bortezomib? Is the enhanced proteasome sensitivity of png-1 compared to ERAD mutants due to accumulation of glycosylated ERAD substrates in cytosol, or are the ERAD mutants simply leaker? i.e. which phenotype is epistatic?

3) What happens in the intestine in skn1-a? It is stated in the manuscript that SKN-1C is expressed in the intestine. Does this provide SKN-1A function in the intestine?

4) The authors make the comment, "We generated similar transgenes to express tagged full length and truncated SKN-1C, but did not observe any effect of proteasome disruption (data not shown)." What exactly is meant by this? Was SKN-1C not expressed under either condition (-/+ proteasome inhibition), or was SKN-1C equally expressed under both conditions?

5) Can the authors please comment on whether the cleavage site reported by Radhakrishnan et al. is in a region that is conserved in SKN-1A? How does the cleavage site in the human protein relate to the N-terminus of the ∆TMD construct?

6) Some of the references are not complete. This includes Arribere, Ayyadevara, Blackwell, Dickinson, Paix, Radhakrishnan 2014 (why is M.S. Brown an author here?), Sha, Zhang 2013.

Reviewer #3:

The transcription factor SKN-1/NRF1 is best known for its roles in oxidative and xenobiotic stress response pathways, but in recent years has become increasingly implicated in protein homeostasis. It responds to translational inhibition, upregulating the stress response, it controls proteasomal gene expression, upregulating target genes in response to proteasomal compromise. Finally it plays an important role in the ER stress response and autophagy regulation. However, the pathways governing these regulatory networks and their potential interconnections are not well understood.

In this work, Ruvkun and co-workers perform genetic screens in C. elegans for loci required for the SKN-1 regulatory response to proteasome inhibition. Interestingly, they identify a number of genes involved in ER associated degradation (sel-1, sel-11, sel-9, png-1), as well as the aspartyl protease ddi-1. They further show that an ER localized isoform of SKN-1a/NRF-1 translocates to the nucleus upon proteasome knockdown, where it upregulates its targets. ERAD knockdown also facilitate SKN-1a stabilization, although without nuclear localization, suggesting that ERAD components control SKN-1 availability from the ER.

The authors focus predominately on characterizing DDI-1, a conserved aspartyl protease whose function is poorly understood. They find that DDI-1 is induced upon proteasomal dysfunction in a SKN-1 dependent manner, and localizes largely to the nucleus. They find that DDI-1 plays a role in proteolytic processing of SKN-1A, which is likely important for its activity.

This is an excellent study that uses the power of genetics to unravel a very topical area, how proteasomal status regulates NRF activity and proteasome surveillance. This work gives key insight into how this might occur via ERAD and proteolytic regulation, providing a plausible link of NRF1 activation with the ER stress response and ERAD. The authors focus on the role of DDI-1 predominately because it is not well studied, thereby lending new insight. Among other things, this study raises the question of how mechanistically ERAD mobilizes SKN-1A from the ER, but perhaps this would be the subject of a whole other study.

I only have a few concerns/questions:

Major: The major question is to what extent does proteasome inhibition link to canonical ERAD signaling?

1) Do other ERAD components, such as CDC-48, play any role in SKN-1 dependent proteasomal activation?

2) Does proteasome compromise trigger the ER stress response, (and thereby SKN-1a activation? i.e. does proteasome knockdown induce UPR or ERAD?

3) Does ddi-1 loss block all transcriptional SKN-1a activity or only targets linked to ERAD and proteasome perturbation?

4) Does ddi-1 play any role in ERAD? Is it required to degrade an ERAD substrate?

https://doi.org/10.7554/eLife.17721.015

Author response

Reviewer #1:

In this body of work Ruvkun and colleagues begin to query proteasome regulation using elegant genetics and the nematode C. elegans. In this data packed manuscript they outline the role of DDI-1, an aspartic protease, that moves to the nucleus under conditions of proteasome stress. The protease domain is more than likely required to regulate a single isoform of skn-1, thereby regulating its nuclear activities. Throughout there is great genetic interaction studies between ddi-1, son-1 and components of the ERAD pathway (which are required to liberate skn-1 from the ER). There is a cornucopia of findings throughout and almost every stone has been turned.

Minor comments:

The western bot shown in Figure 4A is blown out. It’s ok to show it, but the authors should also include a lighter exposed one.

We have added a shorter exposure of the same blot to Figure 4A.

It would also help the reader to include a model describing skn-1 and ddi-1 under static vs. proteasome stress.

We have added a model as a figure supplement to the final figure.

The induction of rapt is followed by GFP reporters. Does the endogenous gene also show similar regulation?

Endogenous rpt-3 has been shown by the Blackwell lab to be induced in a skn-1-dependent manner in response to proteasome disruption (Li et al, 2011). This was one of the reasons we chose to use the rpt-3::gfp reporter. We have added a reference to this point in the results section.

Lastly, the movement of ddi-1 to the nucleus is shown during proteasome stress. Do other stresses also regulate its movement?

This is an interesting question – but we think addressing this properly requires a separate study. We are planning to use the reagents we have generated (DDI-1 and SKN-1A fusion proteins) to perform a more comprehensive survey of the stresses that stimulate DDI-1 to enter the nucleus and cleave SKN-1A.

Reviewer #2:

[…] I have several comments, which are all modest in nature:

1) At the outset of Results, it should be explained what pbs-5(mg502) is, and what is the rationale for its use. I had to do a google search to figure out it is a mutation in a 20S proteasome subunit.

We have clarified this section of the results. We now point out that pbs-5 encodes the beta 5 20S proteasome subunit of C. elegans. And we now indicate that we used this allele for our screen because it causes skn-1 dependent activation of rpt-3::gfp, but otherwise does not affect viability. (Aside – pbs-5(mg502) is one of ~20 proteasome subunit mutations we isolated in genetic screens for constitutive activation of rpt-3::gfp, The other proteasome mutations cause lethality when skn-1 is inactivated by RNAi. Using any of these other mutant strains would have made our screen technically much more challenging, since failure to activate skn-1 would cause lethality).

2) What happens in a png-1 ERAD double mutant in terms of growth in bortezomib? Is the enhanced proteasome sensitivity of png-1 compared to ERAD mutants due to accumulation of glycosylated ERAD substrates in cytosol, or are the ERAD mutants simply leaker? i.e. which phenotype is epistatic?

This is an important question, but we think one best left as part of a future study that more fully addresses the role of ERAD and PNG-1 in SKN-1A activation. (Aside – we have only done the experiment once, and replicates will be needed to confirm, but it appears that the png-1 phenotype is epistatic in png-1; sel-1 double mutants. i.e. the sensitivity of the png-1 mutant is probably not due to accumulation of glycosylated ERAD substrates in the cytoplasm.)

3) What happens in the intestine in skn1-a? It is stated in the manuscript that SKN-1C is expressed in the intestine. Does this provide SKN-1A function in the intestine?

Our experiments with rpt-3::gfp suggest that in the context of proteasome dysfunction caused by RNAi or bortezomib treatment, SKN-1B and SKN-1C do not provide function. In skn-1a mutant animals we have not observed activation of rpt-3::gfp in any tissue, including the intestine, where skn-1C is expressed. We have added a sentence to the results to emphasize this point. One interpretation of our data may be that SKN-1A and SKN-1C (even though they have the same DNA binding domain) are regulated by distinct mechanisms. Indeed, as SKN-1A is the only SKN-1 isoform with a transmembrane domain it is unlikely that ERAD and DDI-1 play a role in SKN-1C activation. This may be analogous to the different mechanisms regulating Nrf1 and Nrf2 in mammals. A full answer to this question will require a deeper understanding of the shared and distinct molecular mechanisms that govern activation of SKN-1A and SKN-1C.

4) The authors make the comment, "We generated similar transgenes to express tagged full length and truncated SKN-1C, but did not observe any effect of proteasome disruption (data not shown)." What exactly is meant by this? Was SKN-1C not expressed under either condition (-/+ proteasome inhibition), or was SKN-1C equally expressed under both conditions?

SKN-1C::GFP expression was not detectable under either condition. We admit that this negative result in itself is somewhat weak, but we think it is important when taken together with the genetic requirement/sufficiency of skn-1a. We favor keeping this sentence in the manuscript – this result (and the existence of the skn-1c::gfp transgenes) should be of interest to SKN-1 aficionados.

5) Can the authors please comment on whether the cleavage site reported by Radhakrishnan et al. is in a region that is conserved in SKN-1A? How does the cleavage site in the human protein relate to the N-terminus of the ∆TMD construct?

The sequence conservation between SKN-1A and Nrf1 is restricted to the DNA binding domain near the C-terminus of the protein; the primary sequence of the cleavage site (AAs 99-108 of Nrf1) does not appear to be conserved. Even within nematodes, the N-terminus of SKN-1A is not highly conserved – although there are two relatively well conserved stretches at AA 122-135 and AA 155-167.

The size (~20kD) of the N-terminal HA::SKN-1A fragment that is present in wild type animals treated with bortezomib, suggests that the cleavage site is somewhere in the vicinity of AAs 140-180 of SKN-1A. This putative cleavage site is some distance from the TMD at AAs 39-59, so we think that the failure to cleave ∆TMD SKN-1A is not a consequence of disrupting the cleavage site. We are working to map the cleavage site using HA::SKN-1A::GFP transgenes with small deletions within AAs 130-180. Future analysis of cleavage-resistant SKN-1A mutants will be needed to clarify the relationship between ER-trafficking and cleavage, and whether there are conserved features between the Nrf1 and SKN-1A cleavage sites.

We have added sentences to the discussion to point out the lack of conservation of the Nrf1 cleavage site and the presence of potential conserved SKN-1A cleavage site(s) in nematode Nrf1/SKN-1A orthologues.

6) Some of the references are not complete. This includes Arribere, Ayyadevara, Blackwell, Dickinson, Paix, Radhakrishnan 2014 (why is M.S. Brown an author here?), Sha, Zhang 2013.

We have corrected the mistakes in the references.

Reviewer #3:

[…] I only have a few concerns/questions:

Major: The major question is to what extent does proteasome inhibition link to canonical ERAD signaling?

This is an important question, but considerable work will be required to tease out the interaction between SKN-1A, ERAD, the UPR and possibly other stress response pathways. We agree with the reviewer’s suggestion above that this would be best addressed by a separate study.

1) Do other ERAD components, such as CDC-48, play any role in SKN-1 dependent proteasomal activation?

Yes, but it’s complicated. C. elegans has two near-identical genes encoding CDC-48 (cdc-48.1 and cdc-48.2) which appear to be redundant – this might explain why no cdc-48 alleles were isolated in our mutagenesis screen. RNAi of either gene apparently silences both paralogs and is lethal. cdc-48.1/2 RNAi of wild type animals causes mild activation of rpt-3::gfp, and cdc-48 RNAi of pbs-5(nic67) mutants causes a mild reduction in rpt-3::gfp expression. RNAi-mediated silencing of the cdc-48 cofactors ufd-1 or npl-4 each cause activation of rpt-3::gfp in wild type animals. Overall these data suggest that the CDC-48/UFD-1/NPL-4 complex may play a role as both a positive and negative regulator of SKN-1A, and more work will be needed to clarify its role.

2) Does proteasome compromise trigger the ER stress response, (and thereby SKN-1a activation? i.e. does proteasome knockdown induce UPR or ERAD?

There is evidence that the UPR is activated by proteasome dysfunction in C. elegans; e.g. Proteasome RNAi induces expression of hsp-4::gfp, a UPR reporter that is regulated by XBP-1. Since the UPR is thought to regulate ERAD, it is reasonable to suspect that the UPR could influence, at least under some circumstances, the activation of SKN-1A. We think testing this is beyond the scope of this study but would be worth doing in future.

3) Does ddi-1 loss block all transcriptional SKN-1a activity or only targets linked to ERAD and proteasome perturbation?

We do not know yet. We plan to perform RNAseq and SKN-1A ChIP experiments to examine this.

4) Does ddi-1 play any role in ERAD? Is it required to degrade an ERAD substrate?

Constitutive degradation of SKN-1A requires the ERAD factors SEL-1 and SEL-11, but does not require DDI-1, so this is at least one ERAD substrate that is still degraded in ddi-1 mutants. Relatively little is known about endogenous ERAD substrates in C. elegans, so time-consuming experiments to find and characterize such ERAD substrates would be required to address this question further.

Related to both this point and point (1) above, it would be interesting to determine which (if any) extraproteasomal ubiquitin receptors participate in ERAD in C. elegans, and whether these affect SKN-1A degradation or activation. Again, this is best the focus for a future study.

https://doi.org/10.7554/eLife.17721.016

Article and author information

Author details

  1. Nicolas J Lehrbach

    1. Department of Molecular Biology, Massachusetts General Hospital, Boston, United States
    2. Department of Genetics, Harvard Medical School, Boston, United States
    Contribution
    NJL, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  2. Gary Ruvkun

    1. Department of Molecular Biology, Massachusetts General Hospital, Boston, United States
    2. Department of Genetics, Harvard Medical School, Boston, United States
    Contribution
    GR, Conception and design, Analysis and interpretation of data, Drafting or revising the article
    For correspondence
    ruvkun@molbio.mgh.harvard.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7473-8484

Funding

National Institutes of Health (R01 AG016636)

  • Gary Ruvkun

Human Frontier Science Program (Long Term Fellowship)

  • Nicolas J Lehrbach

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We would like to thank Ulandt Kim for high-throughput sequencing. We thank Fei Ji and Ruslan Sadreyev for bioinformatics advice. We thank Fred Goldberg and Keith Blackwell and members of their labs for useful discussions. This work was supported by an NIH R01 grant to GR [R01 AG016636]. NL was supported by a Human Frontier Science Program Postdoctoral Fellowship.

Reviewing Editor

  1. Andrew Dillin, Howard Hughes Medical Institute, University of California, Berkeley, United States

Publication history

  1. Received: May 11, 2016
  2. Accepted: July 19, 2016
  3. Version of Record published: August 16, 2016 (version 1)

Copyright

© 2016, Lehrbach et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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